A recombinant lactobacillus that simultaneously expresses Clostridium perfringens α, β2, ε, β1 exotoxin and its construction method and application

A technology of Clostridium perfringens and Lactobacillus, which is applied in the field of veterinary medicine, can solve problems such as difficult to provide effective protection, and achieve the effect of preventing Clostridium perfringens infection

Active Publication Date: 2020-12-18
NORTHEAST AGRICULTURAL UNIVERSITY
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, genetically engineered lactic acid bacteria constructed only against the alpha toxin of Clostridium perfringens type A cannot provide effective protection against other serotypes of Clostridium perfringens

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A recombinant lactobacillus that simultaneously expresses Clostridium perfringens α, β2, ε, β1 exotoxin and its construction method and application
  • A recombinant lactobacillus that simultaneously expresses Clostridium perfringens α, β2, ε, β1 exotoxin and its construction method and application
  • A recombinant lactobacillus that simultaneously expresses Clostridium perfringens α, β2, ε, β1 exotoxin and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of Recombinant Lactobacillus Simultaneously Expressing Clostridium perfringens α, β2, ε, β1 Exotoxin

[0035] 1. Detoxification transformation of Clostridium perfringens exotoxin

[0036]Through the bioinformatics analysis of Clostridium perfringens exotoxins α, β1, β2 and ε, the key gene loci that determine the toxicity of each exotoxin were determined, and the PCR method was used to detoxify and transform them into toxoids. Specifically: mutate residues 202-204 of the gene encoding α-toxin to GGC; mutate residues 699-702 of the gene encoding β-toxin (β1, β2) to GGA; The -318 residue is mutated to CCA, and the nucleotide sequences encoding the detoxified Clostridium perfringens exotoxin α, β2, ε, and β1 genes are shown in SEQ ID NO.5-8 respectively, and the encoded The amino acid sequences are shown in SEQ ID NO.1-4 respectively. Then, the PCR products of each toxin-encoding gene were cloned into pMD19-T vector sequentially, using BamH I and Kp...

Embodiment 2

[0042] Example 2 Recombinant lactobacillus immunogenicity detection

[0043] The present invention systematically evaluates the immunogenicity of the recombinant lactobacillus obtained in Example 1 by using BALB / c mice as animal models. The immunization procedure was as follows: the recombinant Lactobacillus pPG612-α-β2-ε-β1 / LP was inoculated by oral gavage, and the amount of bacteria in each BALB / c mouse was 2×10 8 CFU / mL, immunization once every 2 weeks, a total of 3 immunizations. At the same time, non-recombinant Lactobacillus (LP) and PBS were orally inoculated as controls. The levels of sIgA antibodies in feces, vaginal mucus and tears and the levels of IgG antibodies in serum were detected by ELISA method.

[0044] In addition, the challenge protection test was carried out on the immunized animals: the challenge (purified Clostridium perfringens exotoxin) test was carried out 15 days after the third immunization, and each mouse was intraperitoneally injected with 2 ti...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a recombinant lactobacillus for simultaneously expressing clostridium perfringens alpha, beta 2, epsilon and beta 1 exotoxin, a construction method of the recombinant lactobacillus and an application. The recombinant lactobacillus takes the clostridium perfringens alpha, beta 1, beta 2 and epsilon exotoxin reconstructed from gene levels in a toxicity removal manner as immunogen, the reconstructed exotoxin completely loses pathogenicity and still retains immunogenicity, and the lactobacillus serves as a safe living bacterium carrier for transmitting and expressing immunizing antigens, so that the recombinant lactobacillus capable of simultaneously expressing clostridium perfringens alpha, beta 2, epsilon and beta 1 exotoxin is acquired, and a gene engineering lactobacillus multi-valence subunit toxin oral immune preparation capable of effectively preventing infection of five serotype clostridium perfringens is prevented. The recombinant lactobacillus provides effective technical means for preventing infection of the clostridium perfringens.

Description

technical field [0001] The present invention relates to a recombinant lactic acid bacterium obtained by means of genetic engineering, in particular to a recombinant lactic acid bacterium simultaneously expressing Clostridium perfringens α, β2, ε, β1 exotoxin, and also relates to the construction of the strain and its preparation Use in Clostridium perfringens vaccines. The invention belongs to the technical field of veterinary drugs. Background technique [0002] Clostridium perfringens (Clostridium perfringens) is a typical intestinal pathogenic bacteria, which mainly causes diseases such as necrotic enteritis, enterotoxemia and traumatic gas gangrene in cattle and sheep, and is harmful to the cattle and sheep breeding industry. One of the main bacterial infectious diseases developed. The pathogenic factors of Clostridium perfringens are various exotoxins secreted by it, among which α, β (β1, β2), ε and ι are the most important exotoxins. Clostridium membranes are divide...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/74A61K39/116A61K39/08A61P31/04C12R1/225
CPCA61K39/08A61K2039/70C07K14/33C12N15/74
Inventor 徐义刚王丽李一经唐丽杰乔薪瑗
Owner NORTHEAST AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap